The present invention relates to a micropipette superior in droplet-volume controllability and productivity and preferably used to line and fix micro-volume droplets at a high density for applications such as manufacturing of DNA chips. The present invention further relates to a dispenser using micropipette.
The genetic-structure analyzing method has been remarkably advancing recently, and many genetic structures, including structures of human genes, have been clarified. To analyze the above genetic structures, a DNA chip is used in which thousands to tens of thousands or more of different types of DNA pieces are lined and fixed as microspots on a substrate, such as a microscope slide glass.
As methods for forming microspots in manufacturing the DNA chip, the QUILL method, the pin-and-ring method, and the spring pin method are widely used. Even when any method is used, it is necessary to decrease the fluctuation of volumes and shapes of microspots and keep the distance between microspots constant. Moreover, it is greatly desired that a new method exhibiting superior shape controllability and productivity of microspots is developed to further increase the density.
In this case, the QUILL method is a method for forming a microspot by storing samples in a concave portion formed at the tip of a pin, and making the pin tip contact a substrate, thereby moving the samples in the concave portion onto the substrate. However, there is a problem with respect to durability in that the pin tip is deformed or damaged due to the contact with a substrate, or a problem in that cross contamination easily occurs due to imperfect cleaning of the samples stored in the concave portion.
The pin and ring method is a method for forming spots on a substrate by reserving a sample solution in a microplate with a ring and thereafter catching the sample in the ring with the tip of a pin so that the solution passes through the ring. However, the number of types of samples that can be reserved at one time depends on the number of rings, which has been limited so far. Therefore, to form microspots of thousands to tens of thousands of types of samples, hundreds to thousands of cleaning and drying steps are also necessary. Thus, productivity is not as high as would be desired.
The spring pin method is a method for forming microspots by pressing a sample attached to the tip of a pin against a substrate, thereby moving the sample onto the substrate, in which pin and substrate damage are moderated by a double-pin structure having a built-in spring to spout the sample. However, only one-time spotting can be performed by one-time reservation. Therefore, the method is inferior in productivity.
Furthermore, with these conventional microspot-forming methods, because each sample solution is carried onto a substrate while it is exposed to the atmosphere, trouble occurs in that the sample is dried while it is carried and spotting cannot be performed. Therefore, a problem occurs in that a very expensive sample solution cannot be efficiently used.
Furthermore, a method for performing spotting by using the so-called ink-jet system practically used for a printer was studied. However, forming thousands to ten thousands of samples in separate channels has many problems from viewpoints of size and cost. Moreover, in case of the ink-jet system, it is necessary to fill a pump with samples without any bubbles before spotting. It is necessary to use much of the sample to fill the pump and, therefore, sample use efficiency is inferior. Furthermore, it is better for bubble discharge that a liquid moves through a channel including a pump chamber, at a high speed, thereby being agitated in the channel. Thus, when a delicate DNA solution is used as a sample, DNA may be damaged.
The present invention has been made to solve the above problems, and its object is to provide a micropipette making it possible to form microspots at a high accuracy and a high speed and to provide a dispenser having superior productivity using the micropipette which is capable of forming microspots by efficiently dispensing hundreds to tens of thousands of different samples at one time.
The present invention provides a micropipette comprising at least one substrate, an inlet port through which a sample is delivered from the outside, formed on the at least one substrate, a cavity into which the sample is poured and which is filled with the sample, and an injection port for expelling the sample formed on the at least one substrate. The substrate for forming the cavity is made of ceramics, at least one wall face of the substrate is provided with a piezoelectric/electrostrictive element, and the sample moves as a laminar flow in the cavity, wherein volumes of the cavity are changed by driving the piezoelectric/electrostrictive element, and a certain amount of the sample in the cavity is expelled from the injection port.
Because a micropipette of the present invention uses the above structure, a very small amount of a liquid is expelled through an injection port corresponding to each time a piezoelectric/electrostrictive element is driven and the volume of the liquid is very small and constant. The driving cycle can correspond to a high frequency by using the piezoelectric/electrostrictive element, and the time required for injection is also decreased. Moreover, because a sample moves in a closed space before the sample is expelled after it is delivered, the sample is not dried during that period. Furthermore, because the substrate can be compactly formed, it is possible to shorten the channel through which a sample moves and reduce the deterioration of use efficiency due to the attachment of the sample to the channel wall.
According to the present invention, it is preferable to previously fill a cavity with a displacement liquid, such as a buffer solution or physiologic saline solution, and then to deliver the sample into the cavity through the inlet port while laminar-flow-replacing the displacement liquid with the sample, and thereafter expel the sample in the cavity through an injection port by driving a piezoelectric/electrostrictive element. It is possible to control the completion of the laminar flow-replacing step, that is, the replacement time, by previously obtaining the velocity and the volume of the sample. However, it is more preferable to determine the end of the laminar flow-replacement by detecting the change of fluid characteristics in the cavity. Moreover, it is permitted to laminar-flow-replace a displacement liquid in the cavity with the sample from the inlet port while driving the piezoelectric/electrostrictive element. By previously securely replacing the inside of a cavity with an inexpensive replacement solution and then laminar-flow-replacing the inexpensive solution with an expensive sample, it is possible to completely prevent miss-injection from occurring and efficiently expel the expensive sample.
Moreover, according to the present invention, it is preferable to previously fill a cavity with a replacement solution such as a buffer solution or physiologic saline solution, and then to deliver a sample into the cavity through the inlet port while replacing the replacement solution with the sample, detect the end of the replacement by detecting the change of fluid characteristics in the cavity, and thereafter expel the sample in the cavity through an injection port by driving a piezoelectric/electrostrictive element. By detecting the change of fluid characteristics in the cavity and thereby determining the completion of replacement, it is possible to easily distinguish between the portion where the sample mixes with the replacement solution, and the portion where they do not mix with each other, and accurately clarify the portions even if they slightly mix in the channel. Therefore, it is possible to decrease the quantity of the sample mixed with the replacement solution that must be purged and improve the use efficiency of the sample.
Moreover, it is preferable to determine the change of fluid characteristics in the cavity by applying a voltage for exciting vibrations to the piezoelectric/electrostrictive element, and detecting the change of electric constants due to the vibrations. Thus, it is unnecessary to set a special detection element, and highly accurate yet inexpensive detection is realized.
According to the present invention, it is preferable that a sample inlet port, cavity, a sample injection port, and piezoelectric/electrostrictive element are formed at a plurality of places in one substrate. It is also preferred that a plurality of units, each of which includes a sample inlet port, a cavity, a sample injection port, and a piezoelectric/electrostrictive element formed in a substrate, and fixed to a fixing jig. Or, it is preferred to include three types of portions, such as a combination of a cavity and a piezoelectric/electrostrictive element, a sample inlet port, and a sample injection port are separately formed on at least two types of substrates and joined to each other. Alternatively, it is preferable to provide at least a cavity and a piezoelectric/electrostrictive element formed in the above substrate, and a unit formed by joining the above substrate or more, to one substrate on which one of either of a sample inlet port and a sample injection port are formed, and where the one unit, or more, are fixed and integrated.
Because each portion is formed at a plurality of places in one substrate, it is possible to compactly arrange the portions, form injection ports at a high accuracy and a high density, and expel a plurality of types of samples at the same time. By fixing a plurality of units, in each of which one portion is formed, in one substrate to constitute the whole, each substrate is easily manufactured and the yield is improved. Moreover, by joining at least two substrates, on each of which portions are formed as the whole, the range for selecting materials for the substrate is widened and it is possible to select an optimum material for each portion. Moreover, the yield of elements can be improved, the accuracy of the injection port can be improved, injection ports can be arranged at a high density, and a plurality of types of samples can be expelled at the same time.
Furthermore, it is preferable that the substrate is flat and injection ports of samples are formed on a side face or a major surface of the substrate, or that the substrate is flat, and injection ports of samples are formed on one of the opposing major surfaces of the substrate, and inlet ports of samples are formed on the other major surface of the substrate. By forming the substrate to be flat, the substrate can be manufactured by the green sheet lamination technique described later, and the whole becomes thin and compact. When injection ports are formed on a major surface of a substrate, it is possible to set the substrate in parallel with a flat plate on which injection ports are formed and easily keep the injection distance of droplets constant, and shapes of droplets are stabilized. Moreover, when injection ports are formed on the side face of a substrate, it is possible to longitudinally arrange flat substrates and thereby easily increase the density of the injection ports. Furthermore, by forming an inlet port and an injection port on opposite major surfaces, the length of the channel extending from the inlet port up to the injection port requires almost only the thickness of the flat plate, the channel of a sample solution is shortened and becomes simple, the frequency of the trouble that bubbles are caught in the channel to cause miss-injection can be decreased, and moreover, the sample use efficiency is improved.
Furthermore, it is permitted that two or more sample inlet ports are connected to one cavity. In case of this structure, it is possible to securely fill the cavity with samples by sucking or ejecting samples or a replacement solution through a plurality of inlet ports by adjusting the timing.
Furthermore, according to the present invention, it is preferable that a substrate in or on which a cavity and a piezoelectric/electrostrictive element are formed is made of zirconium ceramics, or that each substrate is made of zirconium ceramics. It is preferable that these substrates are manufactured by the green-sheet laminating and sintering method. Zirconia, is suitable and particularly stabilized zirconia and partially stabilized zirconia, because they have a large mechanical strength, a high toughness, a large durability to an acid/alkaline solution, and a small reactivity with piezoelectric films or electrode materials. Moreover, it is permitted that a substrate on which at least one inlet port and one injection port are formed is made of a metal or resin having superior formability characteristics and low cost.
Furthermore, a piezoelectric/electrostrictive film is preferably used for the piezoelectric/electrostrictive element because it is mainly made of lead zirconate, lead titanate, and lead magnesium niobate. Thereby, it has a high electromechanical coupling factor, a high piezoelectric constant, a small reactivity with the zirconia substrate when the piezoelectric film is sintered, and a stable composition.
Furthermore, the present invention provides a dispenser using a plurality of micropipettes respectively formed so that inlet ports through which a sample is delivered from the outside, cavities to be filled with the sample, and injection ports for expelling the sample are formed on at least one substrate. A piezoelectric/electrostrictive element is provided for at least one wall surface of the substrate for forming the cavities, and the sample moves as a laminar flow in the cavity, wherein the injection ports are vertically and horizontally lined and arranged, and different types of solution samples are injected from the injection ports.
Furthermore, the present invention provides a dispenser using a plurality of micropipettes respectively formed so that inlet ports through which a sample is delivered from the outside, a cavity into which the sample is poured and which is be filled with the sample, and injection ports for expelling the sample are formed on at least one substrate. The substrate forming the cavity is made of ceramics, and the substrate has a piezoelectric/electrostrictive element on at least one wall surface. The cavity is previously filled with a displacement solution, then the sample is poured into the cavity through the inlet ports while replacing a displacement solution with the sample. Completion of sample replacement in the cavity is determined by detecting the change of fluid characteristics in the cavity. Thereafter, the volume of the cavity is changed by driving the piezoelectric/electrostrictive element, and a certain amount of the sample in the cavity is expelled through the injection ports, which are vertically and horizontally lined and arranged, and different types of solution samples are expelled from the injection ports.
These dispensers make it possible to supply many types of samples at the same time by using a plurality of micropipettes and easily replace a locally-defective pipette with a new one. Moreover, because injection ports are vertically and horizontally lined and arranged, each of the above dispensers is preferably adopted when two-dimensionally lined and fixed microspots like a DNA chip are necessary.
It is preferable that each of these dispensers is provided with a mechanism in which cartridges separately filled with different types of solution samples are set to sample inlet ports to deliver different solution samples through inlet ports in order to improve the sample use efficiency. Moreover, it is preferable that each dispenser is provided with a mechanism in which a cartridge filled with a water-soluble solvent or organic solvent is set to each sample inlet port to clean the space from inlet ports up to injection ports formed in the substrate in order to expel thousands to tens of thousands of highly pure DNA pieces to very small spots without contamination.
Moreover, it is preferable that each of the dispensers has a different-directional-flying-droplet shielding plate made of a thin plate having a hole coaxial with an injection port outside the injection port. Thus, even if the expelling direction of an injection droplet is deviated, the droplet does not reach a substrate. Therefore, it is possible to prevent that the troubles of a spotting position being shifted or a spot mixing with an adjacent spot.